The present invention is directed to a method for treating subjects having an angiogenesis-related disease by administering neomycin or an analogue thereof. The present invention is also directed to a pharmaceutical composition comprising (a) neomycin or an analogue thereof, and, optionally, (b) another anti-angiogenic agent or an anti-cancer agent. The invention is further directed to a method for screening neomycin analogues having anti-angiogenic activity. In a preferred embodiment, neomycin is administered to subjects having angiogenesis-related diseases. In other embodiments, neomycin or an analogue thereof is administered with another anti-angiogenic agent. In additional embodiments, neomycin or an analogue thereof is administered with an anti-neoplastic agent to treat subjects having an angiogenesis-related disease which is a cancer.
2.1. Angiogensis
Angiogenesis is the complex process of blood vessel formation. The process involves both biochemical and cellular events, including (1) activation of endothelial cells (ECs) by an angiogenic stimulus; (2) degradation of the extracellular matrix, invasion of the activated ECs into the surrounding tissues, and migration toward the source of the angiogenic stimulus; (3) proliferation and differentiation of ECs to form new blood vessels (See, e.g., Folkman et al., 1991, J. Biol. Chem. 267:10931-10934).
The control of angiogenesis is a highly regulated process involving angiogenic stimulators and inhibitors. In healthy humans and animals, angiogenesis occurs under specific, restricted situations. For example, angiogenesis is normally observed in fetal and embryonal development, development and growth of normal tissues and organs, wound healing, and the formation of the corpus luteum, endometrium and placenta.
2.2. Angiogenesis-Related Diseases
The control of angiogenesis is altered in certain diseases. Many such diseases involve pathological angiogenesis (i.e., inappropriate, excessive or undesired blood vessel formation), which supports the disease state and, in many instances, contributes to the cellular and tissue damage associated with such diseases. Angiogenesis-related diseases (i.e., those involving pathological angiogenesis) are myriad and varied. They include, but are not limited to, various forms of tumors, chronic inflammatory diseases, and neovascularization diseases.
The formation and metastasis of tumors involve pathological angiogenesis. Like healthy tissues, tumors require blood vessels in order to provide nutrients and oxygen and remove cellular wastes. Thus, pathological angiogenesis is critical to the growth and expansion of tumors. Tumors in which angiogenesis is important include solid tumors as well as benign tumors such as acoustic neuroma, neurofibroma, trachoma and pyogenic granulomas.
Pathological angiogenesis also plays an important role in tumor metastasis. Pathological angiogenesis is important in two aspects. In one, the formation of blood vessels in tumors allows tumor cells to enter the blood stream and to circulate throughout the body. In the other, angiogenesis supports the formation and growth of new tumors seeded by tumor cells that have left the primary site.
Pathological angiogenesis is also associated with certain blood-borne tumors such as leukemias, and various acute or chronic neoplastic diseases of the bone marrow. It is believed that pathological angiogenesis plays a role in the bone marrow abnormalities that give rise to such leukemia-like tumors.
Pathological angiogenesis also plays a prominent role in various chronic inflammatory diseases such as inflammatory bowel diseases, psoriasis, sarcoidosis and rheumatoid arthritis. The chronic inflammation that occurs in such diseases depends on continuous formation of capillary sprouts in the diseased tissue to maintain an influx of inflammatory cells. The influx and presence of the inflammatory cells produce granulomas and thus, maintains the chronic inflammatory state.
For a general discussion of the role of angiogenesis in angiogenesis-related diseases see the following references: Moses et al., 1991, BioTechol. 9:630-633; Leek et al., 1994, J. Leuko. Biol. 56:423-435; and Beck et al., 1997, FASEB J. 11:365-373.
2.3. Angiogenic Factors and their Actions
Both normal and pathological angiogenesis apparently require action by one or more angiogenic factors. Many such factors have been identified. They include angiogenin (ANG), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF), epidermal growth factor (EGF), tumor necrosis factor-alpha (TNF-xcex1), tumor growth factor-alpha (TGF-xcex1), and tumor growth factor-beta (TGF-xcex2).
There has not been a complete elucidation of the mechanism(s) by which angiogenic factors induce the various biochemical and cellular events of angiogenesis. However, much is known regarding the action of angiogenin in inducing angiogenesis, which may at least partially model the angiogenic action of other angiogenic factors.
Angiogenin was first isolated from tumor-conditioned culture medium as a result of a search for tumor angiogenic factors (Fett et al., 1985, Biochemistry 24:5480-5486). This search was based on the hypothesis that tumors will not grow beyond a minuscule size unless they are supplied with new blood vessels to provide nutrients and facilitate gas exchange (Folkman, J., 1971, N. Engl. J. Med. 285:1182-1186). Tumors elicit the formation of new blood vessels by secreting angiogenesis factors. Angiogenin has been shown to be a potent inducer of angiogenesis (Hu et al., 1998, in Human Cytokines, Handbook for Basic and Clinical Research, Vol. III, ed. Aggarwal, B. B. pp. 67-91, Blackwell Sciences, Inc., Maldan, Mass.). It induces the formation of new blood vessels in the chorioallantoic membrane (CAM) of chick embryos, and in the cornea and meniscus of the knee of rabbits (Fett et al., 1985, Biochemistry 24:5480-5486, King et al., 1991, J. Bone Joint Surg. 73-B: 587-590).
Angiogenin normally circulates in human plasma at a concentration of about 250 to 360 ng/ml (Blaser et al., 1993, Eur. J. Clin. Chem. Clin. Biochem. 31: 513-516, Shimoyama et al., 1996, Cancer Res. 56:2703-2706). Plasma angiogenin may promote wound healing when it becomes extravascular, e.g., through trauma. Angiogenin mRNA and protein are elevated in tissues and cells of patients with a variety of tumors (Chopra et al., 1995, Proc. Ann. Meet. Am. Assoc. Cancer Res. 36:A516; Li et al., 1994, J. Path. 172:171-175; and Moroianu et al., 1994, Proc. Natl. Acad Sci. USA 91:1677-1681).
Structure/function studies have shown that angiogenin has a weak but characteristic ribonucleolytic activity (Shapiro et al., 1986, Biochemistry 25:3527-35328). That activity appears to be essential for its angiogenic activity (Shapiro et al., 1989, Biochemistry 28:1726-17329). Compounds that inhibit angiogenin""s ribonucleolytic activity also inhibits its angiogenic activity. Many such compounds have been identified or developed. They include the C-terminal peptides of angiogenin (Rybak et al., 1989, Biochem. Biophys. Res. Comm. 162:535-543), the ribonuclease inhibitor from human placenta (Lee et al., 1988, Biochemistry 27:8545-8553, Shapiro et al., 1987, Proc. Natl. Acad Sci. USA 84:2238-2241) and, more recently, a deoxynucleotide aptamer obtained by exponential enrichment.
Angiogenin apparently must interact with endothelial cells in order to induce angiogenesis. Several such interactions have been identified. Angiogenin binds to actin (Hu et al., 1991, Proc. Natl. Acad. Sci. USA 88:2227-2231, Hu et al., 1993, Proc. Natl. Acad Sci. USA 90:1217-1221) and to a 170 kDa putative receptor (Hu et al., 1997, Proc. Natl. Acad. Sci. USA 94:2204-2209) which are expressed on the surface of endothelial cells growing in dense and sparse culture, respectively. Binding of angiogenin to endothelial cells results in activation of phospholipase C (PLC) (Bicknell et al., 1988, Proc. Natl. Acad Sci. USA 85:5961-5965), endothelial cell migration and invasion (Hu et al., 1994 Proc. Natl. Acad Sci. USA 91:12096-12100), proliferation (Hu et al., 1997, Proc. Natl. Acad. Sci. USA 94:2204-2209), and differentiation (Jimi et al., 1995, Biochem. Biophys. Res. Comm. 211:476-483). A cell binding site on angiogenin has been identified. The site is essential for angiogenic activity and yet encompasses residues not involved in the ribonucleolytic activity (Hallahan et al., 1991, Proc. Natl. Acad Sci. USA 88:2222-2226; Hallahan et al., 1992, Biochemistry, 31:8022-8029). Interference with angiogenin""s interaction with its target cells inhibit its angiogenic activity. For instance, both actin and an anti-actin antibody completely abolishes angiogenin-induced angiogenesis in the CAM of chick embryos (Hu et al., 1993, Proc. Natl. Acad Sci. USA 90:1217-1221). Moreover, administration of actin prevent the growth of transplanted human tumor cells in nude mice (Olson et al., 1995, Proc. Natl. Acad. Sci. USA 92:442-446).
Translocation of angiogenin to the nucleus is apparently essential for angiogenic activity. In the interaction with endothelial cells, angiogenin is internalized and translocated to the nucleus by a process that is lysosome and microtubule independent (Moroianu et al., 1994, Proc. Natl. Acad Sci. USA 91:1677-1681; Moroianu et al., 1994, Biochem. Biophys. Res. Comm. 203:1765-1772; Li et al., 1997, Biochem. Biophys. Res. Comm. 238:305-312). Mutated angiogenins that are incapable of nuclear translocation are also incapable of inducing angiogenesis in the CAM of chick embryos (Moroianu et al., 1994, Proc. Natl. Acad Sci. USA 91:1677-1681). Such mutated angiogenins, however, have full ribonucleolytic activity and can bind to endothelial cells.
While some other angiogenic factors do not necessarily have ribonucleolytic activity, they are internalized and translocated to the nucleus (See Savion et al., 1981, J. Biol. Chem. 256:1149-1154; Bouche et al., 1987, Proc. Natl. Acad. Sci. USA 84:6770-6774; Baldin et al., 1990, EMBO J. 9:1511-1517; Sano et al., 1990, J. Cell. Biol. 110:1417-1426; Quarto et al., 1991, J. Cell. Physiol. 147:311-318). Accordingly, it has been proposed that nuclear translocation is a general pathway for those angiogenic factors that is critical to their angiogenic activity (Moroianu et al., 1994, Proc. Natl. Acad. Sci. USA 91:1677-1681; Vallee et al., 1997, CMLS Cell. Molec. Life Sci. 53:803-815).
2.4. Anti-Angiogenic Agents
The centrality of angiogenesis in the myriad of angiogenesis-related diseases has motivated searches for anti-angiogenic agents (i.e., agents that suppress or inhibit pathological angiogenesis). Such searches typically involve examining the activity of candidate agents with in vivo angiogenesis assay systems. Two well established systems for carrying out such examinations are the CAM assay and the corneal neovascularization assay. These two systems examine an agent""s effect on angiogenic factor-induced capillary formation in the chorioallantoic membrane of chick embryos and the cornea of laboratory animals, respectively (Gimbrone et al., 1974, J. Natl. Cancer Inst. 52:413-427).
Many anti-angiogenic agents have been isolated or developed. They include cartilage-derived factors (Moses et al., 1990, 248:1408-1410; Oikawa et al., 1990, Cancer Lett. 51:181-186); angiostatic steroids (Folkman et al., 1983, Science 221:719-725; Crum et al., 1985, Science 230:1375-1378; Oikawa et al., 1988, Cancer Lett. 43:85-92); and angiostatic vitamin D analogs (Oikawa et al., 1989, Cancer Lett. 48:157-162; Oikawa et al., 1990, Eur. J. Pharmacol. 178:247-50) angiostatin (O""Reilly et al., 1994, Cell 79:315-328), endostatin (O""Reilly et al., 1997, Cell 88:277-285), and verostatin (Pike et al. 1998, J. Exp. Med. 88:2309-2356).
Anti-angiogenic agents that inhibit the angiogenic activity of a specific angiogenic factor, angiogenin, have also been identified or developed. They include monoclonal antibody that binds angiogenin (Fett et al., 1994, Biochem. 33:5421-5427); human placental ribonuclease inhibitor (Shapiro et al., 1987, Proc. Natl. Acad. Sci. USA 84:2238-2241); actin (Hu et al., Proc. Natl. Acad. Sci. USA 90:1217-1221); and synthetic peptides corresponding to the C-terminal region of angiogenin (Ryback et al., 1989, Biochem. Biophy. Res. Comm. 162:535-543).
Anti-angiogenic agents of microbial origin also have been identified. Such agents include anthracycline (Npzaki et al., 1993, J. Antibiot. 46:569-579), 15-deoxyspergualin (Oikawa et al., 1991, J Antibiot. 44:1033-1035), D-penicillamine (Matsubara et al., 1989, J. Clin. Invest. 83:158-167), eponemycin (Oikawa et al., 1991, Biochem. Biophys. Res. Comm. 181:1070-1076), fumagillin (Ingber et al., 1990, Nature 348:555-557), herbimycin A (Oikawa et al., 1989, J. Antibiot. 42:1202-1204), and rapamycin (Akselband et al., 1991, Transplant Proc. 23:2833-2836).
Consistent with the idea that pathological angiogenesis underlies angiogenesis-related diseases, many anti-angiogenic agents have been demonstrated to have beneficial therapeutic activity against such diseases. Various types of tumors have been shown to be susceptible to treatments with anti-angiogenic agents. For example, several anti-angiogenin monoclonal antibodies exhibit significant antitumor activity in preventing or delaying the appearance of several different types of tumor xenografts in athymic mice (Olsen et al., 1994, Cancer Res. 54:4576-4579; Olson et al., 1995, Proc. Natl. Acad. Sci. USA 92:442-446). Actin, an angiogenin antagonist, has been shown to inhibit the establishment of various tumor xenografts in athymic mice (Olson et al., 1995, Proc. Natl. Acad. Sci. USA 92:442-446). Eponemycin inhibits the growth of B 16 melanomas (Sugawara et al., 1990, J Antibiot. 43:8-18). 22-oxa-1 xcex1,25-dihydroxyvitamin D2, a potent angiogenesis inhibitor, has been shown to suppress the growth of autochthonous mammary tumors in rats (Oikawa et al., 1991, Anti-Cancer Drugs 2:475-480). AGM-1470, a synthetic analog of fumagillin, has been shown to inhibit the growth of various types of transplanted tumors in mice (Ingber et al., 1990, Nature 348:555-557).
D-penicillamine, in the presence of copper, suppresses angiogenesis. It has been proposed that that activity accounts for the compound""s efficacy in suppressing the inflammatory symptoms of rheumatoid synovitis, which involve pathological proliferation of small blood vessel in the synovium tissue (Matsubara et al., 1989, J. Clin. Invest. 83:158-167).
2.5. Neomycin
Neomycin is an aminoglycoside antibiotic derived from Streptomyces fradiae. It is bactericidal for many gram-negative and gram-positive organisms. It is in clinical use for oral treatment of enteral infections, to reduce microbe numbers in the colon prior to colon surgery, and orally or in enema form to reduce ammonia-producing bacteria in the treatment of hepatic encephalopathy. Absorption of neomycin from the intestinal tract is relatively poor. The usual oral dose is 4 to 8 Gm in divided doses per day. Neomycin is also administered intramuscularly, using a daily dose of 1 to 6 Gm. Damage to the kidney and the eighth nerve occurs in a significant number of patients when neomycin is given parenterally at a higher dose.
Citation or identification of any reference herein shall not be construed as an admission that such reference is available as prior art to the present invention.
The present invention provides a novel method for treating subjects having an angiogenesis-related disease. The method comprises administering to such subjects neomycin or an analogue thereof In a preferred embodiment, neomycin is administered to a subject having an angiogenesis-related disease. In other embodiments, neomycin or an analogue thereof is administered with other anti-angiogenesis agent(s) to such subjects. In additional embodiments, neomycin or an analogue thereof is administered with an anti-cancer agent to treat a subject having an angiogenesis-related disease which is a cancer.
Angiogenesis-related diseases involve excessive, inappropriate or undesired angiogenesis. Without intending to limit the present invention to any particular theory, it is believed that the disease state of angiogenesis-related diseases requires continuing action by one or more angiogenic factors, and such action requires nuclear translocation of the involved angiogenic factor(s). The present invention is based on the surprising discovery that neomycin and Analogues can inhibit nuclear translocation of angiogenic factors and have anti-angiogenic activity (i.e., inhibit angiogenic factor-induced angiogenesis).
The present invention is illustrated by way of examples that demonstrate the efficacy of neomycin in inhibiting the nuclear translocation of angiogenic factors, suppressing angiogenic factor-induced proliferation of endothelial cells, and inhibiting in vivo angiogenesis induced by certain angiogenic factors.
3.1. Definitions
In order to provide a clear and consistent understanding of the specification and claims, including the scope to be given to a term, the following definitions are given to various terms and abbreviations used herein.